Producing
recombinant virus
particles for
therapeutical means is, besides specifically target cells, purification
and
quantification assays of AAV-2, one intention of the Virus Construction
Kit
provided by the iGEM team Freiburg_Bioware 2010. For obtaining a
modular
toolkit, the complex components of AAV-2 were extracted and redesigned
to match
the iGEM standard. Functional activity was tested in cell culture.

Differing
from the wildtype
AAV-2 genome,
the Helper Free System provided by Stratagene comprises three plasmids
and a
specialized production cell line. AAV-293 cells derived from the HEK
cell line
express the stably integrated E1A and E1B helper proteins for efficient
virus
production. The plasmid containing the inverted terminal repeats (ITRs)
is
encapsidated into the preformed capsids after production of
single-stranded DNA
therefore also known as vectorplasmid (pGOI). Promoter, beta-globin
intron and the hGH terminator signal are flanked by the ITRs and serve
in the
host cell for regulation of transgene expression. In addition to that,
the
plasmid coding for the Rep and Cap proteins (pRC) can be provided in
trans
leading to a layer of specificity due to the fact that the two genes
are not
packaged into the capsid since lacking of the ITRs impairs
encapsidation. Another
advantage of the Helper Free System can be attributed to cotransfection
of
another helper plasmid (pHelper), which provides the necessary proteins
normally obtained by superinfection with helper viruses such as
adenovirus or herpes
simplex virus. These helper genes are required for full viral assembly
by
regulating gene expression of Rep and Cap proteins.

The
iGEM team Freiburg_Bioware
2010 provides
a modular Virus Construction Kit for therapeutical applications,
quantification
assays and purification approaches depending on capsid modifications
and the
gene of interest flanked by the inverted terminal repeats (ITRs. In
order to
produce BioBrick-compatible standardized biological parts, we
reengineered the
plasmids and added new components for gene therapy approaches and
analysis of
biological activity of assembled BioBrick parts. Each element required
for
intact and functional plasmids comprising the ITRs, a promoter, a
putative
enhancer element and the hGH terminator was PCR amplified and fused
together de
novo. As shown
in Figure 1, the
vectorplasmid was
assembled
with the produced BioBricks consisting of the left and right ITR
( BBa_K404100
and BBa_K40410), a promoter (pCMV : BBa_K404102 or phTERT:
BBa_K404106)) , the
beta-globin intron ( BBa_K404107), the gene of interests (fluorescent
proteins
mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30:
( BBa_K404112,
mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator
( BBa_K404108).

Figure
1: Overview of
the theoretical
sequence of each BioBrick provided within the Virus Construction Kit
for an intact and fully functional rAAV genome. The plasmid in the
lowest panel was used for tumor killing in combination with plasmids
coding for modified capsid proteins. More detailed infomartion about
these constructs can be found under ‘Arming: Killing the tumor’ and
‘N-terminal fusion for Targeting’.

Organization
of the recombinant
viral DNA was
modified ensuring several layers of specificity to our systems
including a
tumor-specific promoter and suicide genes encoding prodrug convertases.
In
order to modularize the rAAV sequence, each plasmid element (Figure 1)
was PCR-amplified and
cloned into
the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team
Freiburg_Bioware
2010 performed three site-directed mutagenesis in the gene of interest
TK30 ( BBa_K404109)
and cytosine deaminase ( BBa_K404112) for deletion of PstI and
NgoMIV iGEM site (for further information see the results page of
‘Arming –
Killing the tumor’). Since the inverted terminal repeats (ITRs) are
GC-rich
regions forming T-shaped hairpins during replication, PCR amplification
was not
possible. Hence a cloning strategy was developed by the iGEM team
Freiburg in
order to provide BioBrick-compatible ITRs (see ‘Method Development of
Cloning
Strategy for ITRs’).

In Figure
2 the schematic overview
of the
modularization process can be seen which has been followed to conduct
the
assembly steps required for functional vectorplasmids.

Figure 3: Assembly procedure for
fusion of BioBricks and composite parts to a fully assembled and
functional plasmid coding for your gene of interest. This plasmid can
be cotransfected with two helper plasmids providing protein for
assembly and encapsidating of the rAAV genome (your gene of interest)
into the capsids.

The
iGEM team Freiburg_Bioware
provides two
examples demonstrating the assembly procedure for constructing
vectorplasmids.
The first representative example is the fusion of the BioBrick part beta-globin
to the composite parts containing the 5´ elements of the plasmids,
which are
left ITR and CMV or phTERT promoter, respectively.

As
shown in Figure 3
the theoretical cloning performed
for assembling the BioBricks beta-globin intron
and
leftITR_CMV together
can be observed.

Figure 4:Theoretical
cloning of the
composite part leftITR_CMV to the beta-globin
intron BioBrick
leading to the plasmid leftITR_CMV_beta-globin
intron.

The
plasmids were digested with
both XbaI
and PstI (beta-globin intron: BBa_K404107)
or SpeI and
PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the
preparative gel in Figure
4,
the expected bands could be detected under UV light and the extracted
DNA could
be successfully ligated. Each assembly step for producing BioBrick
intermediates was conducted following the same strategy.

Figure
5: Assembly
intermediate in fusion of the vectorplasmids containing different
promoters. Fusion
of the BioBrick part beta-globin ( BBa_K404107) intron to
the composite
parts leftITR_pCMV and leftITR_phTERT, respectively, was performed
following the BioBrick assembly strategy by digesting the insert with
PstI and XbaI and the vectors with SpeI and PstI. The left lane shows
the expected fragment at around 560 bp which corresponds to the beta-globin
intron fragment, in contrast to the two lanes in the center and on the
right which correspond to linearized plasmids after digesting with
above mentioned iGEM restriction sites. M, GeneRuler DNA ladder mix;
Insert, pSB1C3_beta-globin intron; Vector pCMV,
pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.

Separated
fragments were
extracted using
the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated
with
T4-ligase. After ligation has been carried out, E. coli
XL-1B
cells were
transformed and incubated over night at 37°C. Picking clones from the
transformation plate was performed the following day and DYT medium was
inoculated incubating overnight. Plasmid DNA was isolated and test
digestion
revealed that cloning was successful obtaining the composite part
leftITR_CMV_beta-globin
intron ( BBa_K404117).

Plasmid
production
incorporating all
required elements for transgene expression and genome encapsidation
into empty
viral capsids was performed by fusing the downstream elements
consisting of the
hGH terminator and right ITR to the intermediate part providing the
gene of
interest and the promoter fused to the left ITR. Figure 5
demonstrates the
assembly performed
with pSB1C3_leftITR_phTERT_beta-globin intron_mVenus
and
pSB1C3_hGH_rightITR ( BBa_K404116). The fragment obtained after
digestion on the
left lane fits to the hGH-terminator_rightITR length. The isolated
fragments
were ligated and successful assembly was confirmed by test digestion
obtaining
the vectorplasmid pSB1C3_leftITR_phTERT_beta-globin
intron_mVenus_hGH_rightITR
( BBa_K404124).

Figure 6:
Modularization of
the assembled
vectorplasmid containing the phTERT promoter and mVenus as gene of
interest. Fusion
of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part
to the composite parts pSB1C3_hGH_rightITR was performed following the
BioBrick assembly strategy by digesting the insert with XbaI and PstI
and the vector with SpeI and PstI. The left lane corresponds to
linearized plasmid after digesting with above mentioned iGEM
restriction sites whereas the right lane reveals an intensive band at
around 650 bp confirming the expected size of 657 bp of hGH_rITR. M,
GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin
intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.

Since
cloning does not confirm
biological
activity, we analyzed the plasmids and their functional components, hGH
terminator and beta-globin intron, in cell culture.
Assembled
plasmids
have been cotransfected, using AAV-293 cells, which provide the stable
integrated E1A and E1B genes, with helper plasmids required for capsid
assembly and genome encapsidation (pRC and pHelper) in a molar ratio of
1:1:1
(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA
were
harvested 72 hours post transfection and HT1080 cells transduced with
constant
volumes of viral vectors. 48 hours post infection; transduced cells
expressing
the gene of interest were analyzed by flow cytometry. Facilitating and
demonstrating the analysis of functionality of the assembled plasmid,
mVenus
was used in first place since fluorescent proteins enable facile
visualization
using fluorescent microscopy and flow cytometry analysis.

Qualitative
analysis of mVenus
expression
by fluorescence microscopy was conducted using Axio Observer Z1 showing
that
transduced HT1080 cells and non-transduced cells could be easily
distinguished.
In Figure 6 cells
were excited
with 505nm and fluorescence emission at 536nm was detected. Therefore,
successful
infection of tumor cells by recombinant viral particles carrying the
assembled vectorplasmid
coding for mVenus could be demonstrated.

The
iGEM team Freiburg provides
the hGH
plolyadenylation sequence within the ‘Virus Construction Kit’ due to
the fact
that almost every eukaryotic mRNA is processed at their 3´ and 5´end
except for
histone mRNAs (Millevoi
et al. 2006). Pre-mRNAs
contain
two canonical conserved sequences. First, the polyadenylation signal
“AATAAA”
which is recognized by the multiprotein complex and second the GT-rich
region
(downstream sequence element, DSE) which is located 30 nucleotides
downstream
of the cleavage site. The assembled 3´end-processing machinery cleaves
the mRNA
transcript immediately after a CA-nucleotide therefore defining the
cleavage
site (Danckwardt et al.
2008). Recombinant
vectorplasmids
were engineered containing the inverted terminal repeats (ITRs), a
strong
eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of
interest
with and without the hGH terminator signal. Transduction of HT1080
cells with constant
volume of viral particles containing the vectorplasmids and measuring
mVenus
expression 24 hours post infection by flow cytometry demonstrated that
transgene expression of the constructs lacking the hGH termination
signal is
significantly reduced as shown in Figure
7 and Figure 8
confirming the expected results
that hGH is essential for mRNA processing. The iGEM team
Freiburg_Bioware 2010
therefore suggests using the provided hGH termination signal within the
Virus
Construction Kit for optimal gene expression.

Providing
an element assumed to
be an
enhancer of transgene expression (Nott
et
al. 2003), the iGEM team
Freiburg tested
a beta-globin intron derived from the human beta globin
gene
which can
be fused upstream of the desired gene of interest. The beta-globin
intron
BioBrick consists of a partial chimeric CMV promoter followed by the
intron II
of the beta-globin gene. The 3´end of the intron is
fused to
the first 25
bases of human beta globin gene exon 3. The beta
globin
intron
BioBrick is assumed to enhance eukaryotic gene expression (Nott et al. 2003). Analysis
was conducted as
described for the hGH terminator experiment (see above). As shown in Figure 9
and Figure 10 the
vectorplasmid
missing the beta-globin
intron showed a negligible difference in mVenus expression compared to
viral
genomes containing the beta-globin intron.
Considering these
results and
taking into account that a constant volume of viral particles has been
used for
transduction, the difference between the construct containing and
lacking the
beta-globin intron is minimal. Since packaging efficiency of the AAV-2
decreases with increasing sizes of the insert (Dong
et al. 1996), the iGEM
team
Freiburg_Bioware suggests using the beta-globin intron
in
dependence on
the size of your transgene.

Producing recombinant virus particles for therapeutical applications
is, besides specific cell targeting, purification and quantification
assays of AAV-2, one intention of the Virus Construction Kit provided
by the iGEM team Freiburg_Bioware 2010. For obtaining a modular
toolkit, the complex biological system of the Adeno-associated virus
serotype 2 was examined by an exhaustive literature search.
Subsequently, the essential components for AAV-2 particle production
were extracted and redesigned to match the iGEM standard.
The provided tripartite system is independent of a superinfection
of Adeno- or herpes simplex viruses since the genes encoding the
required helper-proteins are co-transfected. Inside the eukaryotic host
cell, the DNA sequence containing the inverted terminal repeats (ITRs)
is extracted and later encapsidated into the preformed capsids after
production of single-stranded DNA. Consequently, this plasmid is known
as the vector plasmid (pGOI). Promoter, beta-globin intron and the hGH
terminator signal are flanked by the ITRs (ITRs, BBa_K404100 and
BBa_K404101) and regulate transgene expression. The vector plasmid
containing the desired gene of interest is cotransfected with the
RepCap plasmid ( BBa_K404001, BBa_K404102 or BBa_K404103) and the
pHelper plasmid. To obtain the fully assembled vector plasmid, several
assembly steps have to be performed.
After assembly of plasmids containing all required elements, vector
plasmid functionality was confirmed in cell culture. AAV-293 cells
stably expressing the E1A and E1B proteins were transfected with three
plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72 hours
post transfection and the tumor cell line HT1080 was transduced with
the recombinant viral vectors encapsidating the gene of interest mVenus
( BBa_I757008).
In the beginning, the iGEM team Freiburg_Bioware 2010 used a commercial
vector plasmid, pAAV-MCS (Stratagene), to determine whether virus
particle production by AAV-293 cells could be achieved. iGEM RFC 25
restriction enzyme sites were introduced and the fluorescent protein
mVenus was subcloned into this plasmid. Subsequently, this plasmid was
used as a reference and compared to the assembled vector plasmid
pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (abbreviated pSB1C3_mVenus,
BBa_K404119). Fluorescence expression data obtained by flow cytometry
analysis are shown in Figure 11 and Figure 12. Comparing mVenus
expression of the reference plasmid and the pSB1C3_mVenus plasmid
revealed that biological functionality of the reassembled plasmid was
preserved.

Idea
of the modular ‘Virus
Construction
Kit’ is to provide all required elements for producing recombinant,
functional
virus particles delivering encapsidated genes of interest to specific
cells.
First step was to modify and modularize the vectorplasmid comprising
basically
the cis-elements for replication (ITRs), a strong eukaryotic or tissue
specific
promoter (pCMV or phTERT), the gene of interest (fluorescent proteins
or
suicide genes) and the hGH termination signal. Each element was
successfully
cloned and reassembled resulting in functional vectorplasmids
determined by flow
cytometry and fluorescence microscopy analyses. Experiments have been
performed
with mVenus since measurement of fluorescent proteins can be easily
performed
and visualized. Considering the results, the iGEM team Freiburg_Bioware
2010
then tested the construct containing the suicide genes thymidine kinase
and
cytosine deaminase. Further details demonstrating efficient tumor
killing,
using prodrug-activating systems, see results page Arming.

In our terminology the term “RepVP123”
encompasses the
whole AAV2 genome excluding the ITRs. The rep locus
comprises four
proteins related to genome replication while the cap
locus codes for the
proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which
are
required for viral capsid assembly. Source of the RepVP123 BioBrick
supplied
within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the
wild-type
AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In
order
to introduce iGEM standard and additionally enabling the possibility to
modify
the viral capsid via integration of certain motives within the viral
loops 453
and 587 a total of twelve mutations within RepVP123 (see Figure 1)
and additionally two mutations
within the pSB1C3 backbone were performed either by Site-Directed
Mutagenesis
(SDM) or by ordering and cloning of specifically designed gene
sequences matching
the required demands. Modifying the pSB1C3 led to iGEM team
Freiburg_Bioware’s
variant of this backbone, pSB1C3_001.

Figure 3Restriction sites within the wild-type rep
gene sequence, which were removed via cloning of synthetized rep gene
fragment into the plasmid. The red box indicates
the region spanned by the synthetic sequence.

Making
the RepVP123
wild-type compatible with the iGEM standards required the removal of
five
restriction sites (see Figure
1).
This was achieved using site-directed mutagenesis for PstI (position
310) and
PstI (4073). The remaining three iGEM restriction sites EcoRI (1578),
PstI
(1773) and EcoRI (1796) were replaced by a synthetic gene fragment,
since the rep
ORF contained these restriction sites in close proximity to
each other plus
an additional KpnI restriction site which was also not desired (see Figure 2).
This gene fragment was cloned into
the rep gene using HindIII and SwaI, which are
single-cutting
restriction enzymes adjacent to the target area. Additionally, BamHI
(859) and
SalI (1239) were removed, because these enzymes were required for
genetically
inserting the loop modifications in VP123.

In
order to
implement the restriction sites necessary for targeting via loop
insertions,
the gene coding for the VP proteins was modified as well. The
introduction of
these restriction required up to four base mutations in a row, hence it
was
decided to synthesize this gene fragment and replace the wild-type
sequence in RepVP123
as well.

Figure 5Results
for transduction efficiency measured by flow cytometry. Fluorescence is
measured in surviving cells. The tested RepVP123
containing four point mutation to delete iGEM and loop insertion
restriction sites does not show any difference within mVenus expression
compared to the wild-type and therefore can be verified working.

Alongside
to
creating an iGEM compatible plasmid, infectivity of the modified
construct was
tested in cell culture via flow cytometry. Then experiments confirmed
that
single cloning steps did not interfere with natural viral infectivity
at first
(see above). But cloning of the synthesized rep
gene fragment into the
plasmid dramatically reduced transduction efficiency as detected by
flow
cytometry. Scrutinizing each mutation and its potential impact,
suggested that
abolished transduction was related to the mutation, which removed the
KpnI
(1721) site. This site is located within a splice site, which is
crucial for
the Rep proteins, and thus even silent mutations may interfere with
virus
production (see extra topic “Rep proteins”).

Therefore,
additional constructs were designed containing only the synthetic cap
sequence or both, the synthesized sequences plus a re-mutation of KpnI
(1721), in
order to re-establish the wild-type splice site within the rep
ORF. Results
from cell culture obtained via FACS revealed that in fact the poor
results were
related to the KpnI restriction site deletion. Both constructs showed a
transduction efficiency corresponding to the unmodified wild-type RepVP123’s
transduction efficiency.

To
fulfill iGEM requirements all plasmids need to be submitted in pSB1C3,
therefore primers were ordered for amplifying RepVP123
containing all
modifications done so far by PCR and cloning the into pSB1C3. Still,
pSB1C3
contains two restriction sites for SspI and PvuII restriction enzymes
in its
CAT marker. Since these are necessary for cloning ViralBricks in this
vector,
the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM
Headquarters
to implement a new standard for the pSB1C3 backbone which was named
pSB1C3_001.
Both restriction sites interfering with ViralBrick insertions were
mutated to
make SspI and PvuII single-cutters (see method development).

Figure 10Comparison
of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and
PvuII are marked by red boxes.

RepVP123
containing both rep and cap
synthetic gene fragments including
the re-mutation of KpnI and the downstream p5TATA-less promotor was
cloned into
the newly constructed pSB1C3_001. Testing this newly assembled plasmid
in cell
culture revealed unexpected data. Not only did the newly assembled
plasmid work
(see Figure 10), but in comparison to pAAV containing the same RepVP123
construct, pSB1C3_001 showed an about 3 times higher transduction
efficiency.
Although exact reasons are still unknown, these results are probably
related to
the reduced length of pSB1C3_001 compared to the original pAAV plasmid
of
approximately 1000 base pairs.

Shutting-down
the
natural viral tropism is essential for targeting specifically tumor
cells and
not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware
2010
decided to knock-out the viral natural tropism delivered by the heperan
sulfate
proteoglycan-(HSPG) binding site within the viruses 587 loop. The
knock-out was
cloned by designing primers containing the required base exchanges and
performing a SDM. Like performed before, this RepVP123
variant was tested
in cell culture as well and evaluated by flow cytometry. Results show
that
mutation of HSPG-binding motif has severe impact on transduction
efficiency
thus enabling a viral particle carrying this knock-out and additional
targeting
motifs, e.g. within the loops or presented via N-terminal fusion to
bind target
cells’ receptors and therefore infecting target cells at a much higher
rate
compared to unspecific infection of other cell types within an organism
(see Figure 12).

To
quantify
differences in infectivity, the infectious titer of viral particles
built-up of
RepVP123 with and without HSPG binding motif was
determined by qPCR (see
Figure 14) for
different cell
lines. Results show that the implemented HSPG-knock-out verifies
results obtained
from flow cytometry, infectious titers severely compared to RepVP123
with intact HSPG binding motif.

Figure 14Infectious
titers of RepVP123 with and without natural HSPG
binding motif tested in different cell lines via qPCR. Shutting-down
the HSPG binding motif reduces infectious titer in both HT1080 and HeLa
cell lines. For A431 cells, no infectious titer could be detected via
qPCR, which is probably related to poor transduction efficiency of A431
cells.